PROJECT SUMMARY Actin cytoskeleton-based processes play crucial roles in meiosis in mammalian oocytes, including positioning of the meiotic spindle and asymmetric cell division. These events in oocytes are essential for reproductive success, as gene knockouts that impair these processes cause female infertility. The overall goal of the research proposed here is to analyze the roles of key proteins hypothesized to modulate function of the actin cytoskeleton during female meiosis. The hypotheses to be pursued here are based on our published data and on new unpublished data presented here. A second goal of this project is a technical one ? to provide valuable proof-of-principle demonstration of the utility of a novel system for post- translational protein depletion, as an asset to the oocyte biology research community. Standard approaches for protein depletion that are used in oocytes (i.e., knockout, knockdown) have relatively little temporal precision for depletion of the target of interest. In contrast, consider the following example, from study in budding yeast. Chronic depletion of a protein via a null mutation merely produced a slow growth phenotype, whereas depletion at a specific stage of meiosis provided much sharper insights with a much more specific and interesting phenotype. This is the inspiration for the project proposed here. This research will use this same innovative approach ? the auxin-inducible degradation (AID) system ? to gain new insights into the mammalian oocyte's progression through meiosis. We show here that we have the AID system up and running in mouse oocytes. The overall concept for these studies is to express function- altering variants of proteins of interest (dominant-negative [DN] or constitutively-active [CA]) in wild-type oocytes to perturb function of a particular pathway. The AID system allows for degradation of this function- altering protein at different times of meiotic maturation (e.g., M-phase entry [nuclear envelope breakdown], early M-phase, late M-phase, etc.). In essence, this converts oocytes from a mutant state to wild-type at a time of our choosing, allowing temporal specificity in assessing protein function. We will use the AID system to analyze different stages of oocyte meiosis, from entry into M-phase of meiosis I to the conclusion of meiosis II. Thus, this project will be valuable not only for the discoveries it will provide, but also for the demonstration of AID system utility at multiple stages of meiosis. Aim 1 focusses on spindle positioning in meiosis I, and will determine the role of the actin-depolymerization protein cofilin through studies of when and where cofilin is required for spindle positioning. Aim 2 examines meiosis I, metaphase II arrest, and completion of meiosis II, and will test the hypothesis that inappropriate activity of the actin-to- membrane linker protein family known as ERMs impairs spindle function and polar body emission. Taken together, these studies will provide important insights to advance understanding of these crucial actin- dependent processes in oocytes, including leading to future work in in vivo models.